In continuation of our currently funded program, the goal of this work remains the development of capillary electrophoresis as a high throughput, inexpensive tool for DNA sequencing. Under the current program, we have developed replaceable sieving matrices of linear polyacrylamide for DNA sequencing. Initial work involved separation to approximately 350 bases in 1/2 hour. Currently, with new compositions and improved polymerization, we should achieve approximately 500 bases in l hour. One goal of the present proposal is to incorporate this technology into primer walking techniques of sequencing in collaboration with Drs. William Studier, Brookhaven National Laboratory, and Levy Ulanovsky, Weizmann Institute, utilizing the dye-labelled terminator approach. A second collaboration involves the use of the replacement matrices with multiplex sequencing under Dr. George Church, Harvard Medical School. As a second goal, we will continue our work in development of matrices to extend the sequence read range to 700 or greater. In all cases, our strategy will be to replace the matrix after one or several sequence runs in order to remove issues of column lifetime and to create a system of high flexibility. A third goal of the proposed work will be the development of a multicapillary system, incorporating the replaceable matrix. We will design a capillary array port in which injection occurs either simultaneously in all capillaries or sequentially. After the run, the matrix will be blown out and replaced with a new one. We shall develop initially a l0-capillary system, ultimately leading to 50 or more capillaries. In the detection system, we will initially work with a two wavelength-single laser confocal system with a cooled and intensified CCD. This instrument will then be followed by a 2-laser -2-window approach with a CCD for the highest spectral resolution and sensitivity. Finally, we will also explore internal labelling with 4 separate lanes/columns to assess long range sequencing. The ultimate goal of this research is the development of capillary electrophoresis as a high throughput tool for DNA sequencing using primer walking and multiplex sequencing approaches.